We have continued the molecular and functional characterization of the v-raf oncogene and its cellular homologs and are investigating their involvement in the etiology of human cancers. Four genes related to v-raf have now been identified in man: c-raf-1, c-raf-2, Delta-raf, and Gamma-raf. C-raf-2 is a pseudogene in man located on chromosome 4. C-raf-1, the cellular homolog of v-raf, is an active gene located on chromosome 3p25, a site which is specifically altered in small cells lung carcinoma, ovarian carcinoma, and mixed salivary gland tumors. C-raf-1 specifies a protein of 648 amino acids with a calculated molecular weight of 74 kd. The complete c-raf-1 protein was expressed in E. coli using a complete human fetal liver cDNA cloned into an expression vector. We have also demonstrated the normal protein in mouse fibroblasts by immunoblot analyses. The c-raf-1 gene is composed of 17 exons which span over 45 kb of DNA. Northern hybridization analyses with exon and intron-specific c-raf-1 (genomic) probes has allowed us to construct a transcriptional map for the c-raf-1 locus and has demonstrated two alternatively spliced poly(+) mRNAs. One of these mRNAs, containing the most 3' portion of c-raf-1, may be specifically associated with transformation. A second step in the oncogenic activation of c-raf-1 appears to be truncation as all three transforming versions of c-raf-1 are amino-terminally truncated; v-raf with 37 kd, v-mil with 40 kd, and an LTR-inserted and activated c-raf-1 with 50 kd. There are additional raf-related sequences in mouse and human DNA. One of them, isolated from a mouse spleen cDNA library using v-raf, corresponds to an active gene, delta-raf, which is located on human chromosome 7 near the centromere. Another, Gamma-raf, was isolated from a human fetal liver cDNA library using Delta-raf as probe. Homology between Delta-raf and c-raf-1 is 69% for DNA and 74% for amino acid sequences. There are two Delta-raf transcripts of 2.3 and 4.0 kb. We have investigated the tissue-specific expression of c-raf-1, Delta-raf, and gamma-raf and have found that, in contrast to c-raf-1 which is fairly ubuquitious in its expression, transcription of Delta-raf and Gamma-raf shows tissue specificity.